Selenium is an essential micronutrient in the diet of humans and other mammals and many health benefits have been ascribed to this element including preventing cancer, heart disease and other cardiovascular and muscle disorders, inhibiting viral expression, delaying the progression of AIDS in HIV positive patients, slowing the aging process, and having roles in mammalian development, male reproduction and immune function. We proposed previously that the health benefits of selenium are due in large part to the presence of selenium in selenoproteins as the selenium-containing amino acid, selenocysteine (Sec), and since little was known about how Sec was biosynthesized, we undertook a project to elucidate how this amino acid, which is the 21st amino acid in the genetic code, was synthesized and to identify and characterize each of the components involved in its pathway. We established the biosynthetic pathway of Sec in eukaryotes and archaea and are now characterizing the components responsible for its synthesis. We previously generated a conditional knockout mouse that targeted the removal of SECp43, a protein involved in the methylation of Um34 on Sec tRNA. The Um34 methylase is responsible for the final step in the maturation of Sec tRNA and the resulting mature isoform of Sec tRNA has been shown in our laboratory to be responsible for the expression of the subclass of selenoproteins involved in stress-related phenomena in mammals which are designated as stress-related selenoproteins. Knockout of the SECp43 gene is embryonic lethal but its removal in mouse liver has no apparent effect on the animal except that the level of the Sec tRNA population is increased approximately five fold. We are currently characterizing the effect of the liver SECp43 knockout mouse on the Um34 methylase. Furthermore, we generated a knockout mouse that targeted the removal of the selenophosphate synthetase 1 (SPS1) gene in mice. SPS1 was discovered by other investigators and originally described as generating selenophosphate which is the active donor of selenium in Sec biosynthesis. However, we subsequently demonstrated that this protein does not function in selenophosphate biosynthesis and its function is not known. We are therefore characterizing its function by targeting the removal of the SPS1 gene in mice. In addition, our laboratory is collaborating with Dr. B. J. Lee's group in characterizing the function of SPS1 in Drosophila and mammalian cells. These collaborative studies have shown that: 1) the knockdown of SPS1 in Drosophila SL2 cells results in elevated levels of glutamine and causes the formation of megamitochondria;and 2) human SPS1 has five splice variants wherein each variant manifests unique expression patterns, cellular interactions and subcellular locations. In addition, we determined that Um34 methylation on the Sec isoform designated mcmU to form mcmUm requires that the mcmU isoform must be aminoacylated prior to Um34 addition, i.e., the substrate for Um34 methylase is selenocystenyl-tRNA. These findings were established by injecting Xenopus oocytes with synthetic Sec tRNA[Ser]Sec encoding a mutation at position 74, which is the discriminator base of Sec tRNA[Ser]Sec, and cannot be aminoacylated. We observed that this mutant tRNA can be modified at all positions containing modified bases in Sec tRNA[Ser]Sec, including the formation of mcmU, but cannot form Um34. We have also shown that cysteine (Cys) which is normally inserted into protein in response to UGU and UGC codons can be inserted into protein in response to UGA Sec codons, thus replacing Sec with Cys. We demonstrated that thiophosphate was synthesized by selenophosphate synthetase 2 (SPS2) and then the resulting product was donated to the phosphoseryl moiety on Sec tRNA[Ser]Sec. Phosphoseryl-tRNA[Ser]Sec is the intermediate in the biosynthesis of Sec. SPS2 can use sulfide in place of selenide generating thiophosphate that serves to donate sulfur by the enzyme, selenocysteine synthetase, to phosphoseryl-tRNA[Ser]Sec forming cystenyl-tRNA[Ser]Sec. These data provide a novel Sec machinery-based mechanism for the biosynthesis and insertion of Cys into protein and suggest new biological functions for thiophosphate and sulfide in mammals.